The long-term objective of the proposed research is to develop Tanapoxvirus (TPV) replication-competent variants suitable for colorectal cancer therapy in humans. Several novel features of TPV make it an attractive candidate for progression towards this objective. These include the immunological naivet[unreadable] of the potential patient population, its inability to spread through person-to-person contact, and its ability to integrate large (>25 kbp) insertions into its genome. Both rational genetic engineering and random mutation approaches will be employed to generate TPV mutants, which will be tested for their ability to replicate in human colorectal cancer cell lines but not in normal human cell lines. This will be done by constructing TPV mutants with a deleted thymidine kinase gene and 2L gene. To these base constructs we will individually insert the genes for human granulocyte-monocyte colony stimulating factor, human macrophage inflammatory protein 1, and bacterial flagellin. These insertions will enhance the host innate and adaptive anti-tumor immune responses. Additionally, DNA alkylating agents will be used to randomly mutate TPV and the resulting mutants tested for selective replication in human colorectal cancer cell lines and normal human cells. Whatever the source, virus variants/mutants which replicate in human colorectal cancer cell lines but not in normal human cells will then be tested in nude mice for their ability to regress xenografted human colorectal tumors. Another major objective of this proposal is to attract and integrate graduate and undergraduate students in biomedical research. Students will be trained to conceptualize, design and prosecute experiments. This training will also enhance the analytical abilities of students, and add scientists interested in basic biomedical research to the future pool of scientists in this country. Cancer is the result of a heritable change in the cellular program that involves aberrations in gene expression. Viruses, being obligate intracellular parasites at the genetic level, have evolved over millions of years to take advantage of these aberrations for their survival. Not only will this proposal generate TPV mutants which are selectively replication-competent in human colorectal cancer cells and prepare students for basic biomedical research, but the discovery of such mutant viruses offers a unique opportunity to gain insight into the molecular mechanisms involved in cellular differentiation and neoplasia. PUBLIC HEALTH RELEVANCE: Colorectal cancer is a major medical problem in this country and around the world. The purpose of this research proposal is to generate Tanapoxvirus mutants such that these mutants will replicate and destroy cancerous cells, but do not destroy normal cells. In other words, we will have an oncolytic virus for the treatment of colorectal cancer.